Microbiological process for making UK-61,689

ABSTRACT

Actinomadura roseorufa mutants characterized by the ability to produce by fermentation UK-61,689, an acidic polycyclic ether anticoccidial antibiotic previously available only by selective acid hydrolysis of UK-58,852; and Actinomadura roseorufa having the identifying characteristics of ATCC 53,666, ATCC 53,665, ATCC 53,664 and ATCC 53,674.

This is a continuation of application Ser. No. 07/935,673, filed Aug.25, 1992, now abandoned, which is a continuation of application Ser. No.07/506,722, filed Apr. 9, 1990, now abandoned, which is a continuationof application Ser. No. 07/113,563, filed on Oct. 26, 1987, abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a microbiological process for producingUK-61,689, an acidic polycyclic ether anticoccidial antibioticpreviously available only by chemical means. More particularly itrelates to fermentative production of UK-61,689 by cultivatingActinomadura roseorufa having the identifying characteristics of ATCC53,666; and to Actinomadura roseorufa mutants having the identifyingcharacteristics of ATCC 53,665, ATCC 53,664 and ATCC 53,674.

2. Description of Related Art

EP-0169011, published Jan. 22, 1986 describes production of UK-58,852, apolycyclic ether antibiotic produced by cultivation of Actinomaduraroseorufa Huang sp. nov., ATCC 39,697 in an aqueous nutrient mediumunder submerged aerobic conditions.

UK-61,689, a monoglycone acidic polycyclic ether has formula (I),##STR1## Its preparation by the selective acid hydrolysis of UK-58,852,a diglycone polycyclic ether having formula (II), wherein R and R' are H##STR2## is described in British Patent Application No. 8618844, filedAug. 1, 1986. The process described therein comprises acid hydrolysis ofUK-58,852, preferably using 1:1 equivalents of p-toluenesulfonic acidper equivalent of the sodium salt of UK-58,852 in acetonitrile/water assolvent at room temperature.

The preparation of UK-58,852, itself an effective antibiotic, especiallyanticoccidial agent, is described in EP application 169011, publishedJan. 22, 1986. It is produced by submerged aerobic fermentation inaqueous nutrient media of Actinomadura roseorufa Huang sp. nov. ATCC39,697. Also produced in the fermentation with UK-58,852 are two relatedminor components, each of which is antibiotically effective incontrolling coccidiosis. The two minor components, designated asCP-70,228 and CP-70,828, have formula (II), above, wherein R is H and R¹is CH₃ ; and each of R and R¹ is methyl, respectively.

SUMMARY OF THE INVENTION

This invention is concerned with microbiological processes for makingUK-61,689, a valuable acidic polycyclic ether antibiotic and potentanticoccidial, which comprises cultivating mutants of Actinomaduraroseorufa ATCC 53,666 in an aqueous nutrient medium under, preferablysubmerged, aerobic conditions. It is especially concerned with mutantsATCC 53,674 and 53,665 derived from Actinomadura roseorufa ATCC 53,666,which are characterized by their ability to produce UK-61,689 along withUK-58,852 and to a mutant of ATCC 53,665, which is characterized by itsability to produce 61,689 substantially free of UK-58,852, said mutanthaving the identifying characteristics of Actinomadura roseorufa ATCC53,664.

DETAILED DESCRIPTION OF THE INVENTION

The UK-61,689 and UK-58,852 producing microorganisms were obtained bymutation of a new strain of Actinomadura roseorufa, designated FD-27684,(ATCC 53,666), isolated from a soil sample collected in Yamae Village,Kamamoto, Japan. N-methyl-N'-nitro-N-nitrosoguanidine (NTG) was used asmutating agent. Single colonies of the treated microorganism were thenexamined for production of UK-61,689. The general procedure comprisedgrowing ATCC 53,666 in an aqueous nutrient medium under submergedaerobic conditions with shaking at a temperature of 28° C. The choice ofmedium for the growth stage is not critical. A medium consisting ofcerelose (10.0 g), corn starch (5.0 g), corn steep liquor (5.0 g), NZAmine YTT (5.0 g), (registered trademark for enzymatic digest of casein,Humko Sheffield Chemical Co., Inc.), and cobalt chloride (0.002 g) issuspended in one liter of water, pH adjusted to 7.0 with sodiumhydroxide and dispensed (800 ml) to a Fernbach flask. Aftersterilization by autoclaving, flasks are inoculated with a slant growthsuspension or frozen vegetative mycelia, then incubated with agitationon a shaker at about 200 rev/min and a temperature of 28° C. for 8 days.A 50 ml aliquot is then removed, and the mycelia homogenized by a Teflonpestle tissue grinder followed by ultrasonic fragmentation. Thefragmented mycelia were then centrifuged, washed free of medium, thenresuspended in 50 ml of fresh medium in a 300 ml Erlenmeyer flask, andincubated by shaking at 32° C. for two hours after which the cells wereagain centrifuged, washed free of medium with sterile water andsuspended in 50 ml of tris(hydroxymethyl)aminomethane-malate buffer pH9.0. Aliquots of this suspension were then treated with the mutagenicagent NTG at concentrations of 750 mcg to 1500 mcg/ml for one hour on arotary water bath shaker at 250 to 300 rev/min and a temperature of 34°C. After treatment the cells were centrifuged, washed free of themutagen with sterile water and suspended in flasks of fresh growthmedium which were grown by shaking at 32° C. in a cabinet shaker at 200rev/min. After three days the mycelia outgrowth were homogenized andsonicated. Aliquots of the sonicate were serially diluted, plated onto asolid nutrient medium and the plates incubated at 28° C. until thecolony forming units were of sufficient size for transferring to slants.A suitable medium for plates and slants is ATCC Medium No. 172 with N-ZAmine Type A (Humko Sheffield Chemical Co., Inc.) decreased to 1.0 g/l.The inoculated slants were allowed to grow at 28° C. for 10 to 14 daysafter which time they were ready for testing. This was done byinoculating 300 ml Erlenmeyer flasks containing 25 ml of a suitablemedium (one such contains cerelose, 45.0 g; soy flour, 10.0 g; cornsteep liquor, 15.0 g; MnSO₄.H₂ O, 0.1 g; MgSO₄.7H₂ O, 0.1 g; cobaltchloride, 0.002 g; and calcium carbonate, 3.0 g; one liter of water andthe pH of the medium is adjusted to 7.0). After sterilization byautoclaving for 30 minutes at 121° C., the flasks were inoculated withindividual slant growth suspensions and incubated by shaking at 28° C.on a New Brunswick shaker for 7 days. The mutant culture FD-28454 (ATCC53,674) was detected by examining methylisobutyl ketone extracts ofharvested whole broths after spraying developed thin-layerchromatographic plates (silica gel) with vanillin reagent and heating at100° C. for five minutes. The developing system was composed of 9 partschloroform to 1 part methanol which gave Rf values of ˜0.3 for UK-61,689and ˜0.65 for UK-58,852. The mutant culture thus obtained produces amixture of UK-61,689 and UK-58,852. The ratio of UK-61,689/UK-58,852appears to vary somewhat depending upon the conditions of thefermentation. The morphological and cultural characteristics of thethus-obtained mutant are substantially those described herein for A.roseorufa ATCC 53,666. The distinguishing characteristic of this mutantis its ability to produce a mixture of UK-61,689 and UK-58,852 in whichUK-61,689 is the predominant product. Cultivation of the mutant andisolation of antibiotic UK-61,689 may be conducted under conditionssimilar to those employed in previous fermentations yielding polyetherantibiotics. See, for example U.S. Pat. No. 4,361,649. Cultivationpreferably takes place in aqueous nutrient media under preferablysubmerged aerobic conditions with agitation at a temperature of 24° C.to 36° C. Nutrient media useful for cultivation include a source ofassimilable carbon such as sugars, starches and glycerol; a source oforganic nitrogen such as casein, enzymatic digest of casein, soybeanmeal, cotton seed meal, peanut meal, wheat gluten, soy flour, meat mealand fish meal. A source of growth substances such as grain solubles,fish meal, cotton seed meal and yeast extract as well as mineral saltssuch as sodium chloride and calcium carbonate and trace elements such asiron, magnesium, copper, zinc, cobalt and manganese may also be utilizedwith advantageous results. If excessive foaming is encountered duringfermentation, antifoam agents such as vegetable oils or silicones may beadded to the fermentation medium. Aeration of the medium in vessels forsubmerged growth is preferably maintained at the rate of 1/2 to 2volumes of sterile air per volume of fermentation broth per minuteforced into the broth through a sparger. Agitation may be maintained bymeans of agitators generally familiar to those skilled in thefermentation art. The rate of agitation depends on the type of agitatoremployed. A shake flask is usually run at 150 to 300 cycles per minutewhereas a fermenter is usually run at 300 to 1700 revolutions perminute. Aseptic conditions must, of course, be maintained through thetransfer of organism and throughout its growth.

Inoculum for preparation of the antibiotic according to this inventionmay be obtained by employing growth from a slant of the culture or Rouxbottles inoculated with the culture or a thawed mycelia suspension ofthe culture. A solid medium suitable for initial growth of the organismon slants and Roux bottles is ATCC Medium No. 172. The liquid mediumpreviously mentioned in the mutational study is suitable to prepare thevegetative mycelia prior to freezing. The growth may be used toinoculate either shake flasks or inoculum vessels or the inoculumvessels may be seeded from the shake flasks. Maximum growth in shakeflasks is usually reached in 4 to 8 days, whereas inoculum in submergedinoculum vessels will usually be in the most favorable period in 4 to 5days.

The progress of the antibiotic production during fermentation can bemonitored qualitatively by thin-layer chromatography after visualizationby spraying with vanillin reagent as previously described or thedeveloped plate can also be overlayed with brain heart infusion agarseeded with Bacillus subtilis and incubated at 37° C. for 16 hours tovisualize the antibiotics. Thin-layer chromatography is also a usefultool for analyzing the composition of crude and purified materialsextracted from the fermentation broth. A HPLC method employing a 10cm×4.6 mm microbore C-18 column, a 40/200/760 0.01M ammoniumcarbonate/acetonitrile/methanol mobile phase, employing a refractiveindex detector to quantitate the amount of UK-61,689 and co-producedUK-58,852 in fermentation broths.

The antibiotic UK-61,689 produced by the fermentation of the hereindescribed mutants accumulates in the mycelium and in the broth and canbe separated and recovered by extracting the harvested whole unfilteredfermentation broth, i.e., the whole broth, with an organic solvent suchas chloroform, ethyl acetate, methylisobutyl ketone or butanol at thenaturally prevailing pH. Alternatively, to avoid serious emulsionproblems the mycelium is separated and both it and the clarified brothextracted individually with an organic solvent. The solvent extracts arethen concentrated to a thin syrup and pure UK-61,689 obtained bychromatography.

A typical method of separation and recovery of the antibiotic is asfollows: The whole broth from the fermentation of the mutant wasextracted with methylisobutyl ketone. Evaporation of the extract invacuo gave a reddish oil which was dissolved in ethyl acetate and pouredonto a column of silica gel. The silica gel column was then eluted withethyl acetate and the eluates examined by thin-layer chromatography.Fractions containing UK-61,689 were combined and evaporated to dryness.The thus-obtained UK-61,689 can be further purified by crystallizationfrom isopropyl ether.

The UK-61,689 can be recovered from the fermentation in association withthe mycelium by evaporation of the whole broth by known methods,including spray drying, or by separation of the mycelium from the brothby filtration or centrifugation. The mycelial products thus obtainedcomprise UK-61,689 on the surface of the mycelium and in the intersticesthereof rendering the mycelium a useful carrier for UK-61,689.

A single colony isolate, designated FD-28474, of the above-describedmutant FD-28454 (ATCC 53,674) was itself subjected to mutagenesis by NTGaccording to the procedure described above for preparation of FD-28454.This procedure gave rise to a further mutant (FD-28499) which exhibitedthe morphological and cultural characteristics of Actinomadura roseorufaATCC 53,666 and, of course, of the first produced mutant. However, thismutant (FD-28499) differs from mutants FD-28454 and 28474 in that itproduces UK-61,689 substantially free (i.e., <1%) of UK-58,852. MutantFD-28499 is cultivated in the same manner as is the first-describedmutant (FD-28454) and the UK-61,689 recovered from the fermentation aspreviously described.

This last mutant, identified in the culture collection of Pfizer Inc. asFD-28499, mutants FD-28454 and FD-28474, and the starting microorganism,FD-27684, have been deposited under the terms of the Budapest Treaty inthe American Type Culture Collection, Rockville, Md., a recognizeddepository affording permanence of the deposits and ready accessibilitythereto by the public if a patent is granted on this application. Theyhave been given the designations Actinomadura roseorufa ATCC 53,664,ATCC 53,674, ATCC 53,665 and ATCC 53,666, respectively. The deposits areavailable during pendency of this application to one determined by theCommissioner of the United States Patent and Trademark Office to beentitled thereto under 37 CFR 1.14 and 35 USC 122, and in accordancewith foreign patent laws in countries wherein counterparts of thisapplication, or its progeny, are filed. All restrictions on theavailability to the public of the microorganisms deposited will beirrevocably removed upon granting of a patent thereon.

Taxonomic investigations of FD-27684, FD-28474 and FD-28499 were carriedout by L. H. Huang who provided the following descriptions.

Each of the cultures was planted from a slant into ATCC no. 172 brothand grown for four days at 28° C. on a shaker. It was then centrifugedfor 20 minutes, washed three times with sterile distilled water andplanted on media commonly used for identification of members of theActinomycetales.

The cultures were incubated at 28° C. and the results were read atvarying times but most commonly were taken at 14 days. The colors aredescribed in common terminology, but exact colors were determined bycomparisons with color chips from the Color Harmony Manual, Fourthedition. The method of whole-cell amino acid analysis is that describedin Becker et al., Appl. Microbiol., 12,421-423, 1964. Whole-cell sugarswere analyzed by the methods described in Lechevalier, J. Lab. Clin.Med., 71, 934-944, 1968; and in Staneck and Roberts, Appl. Microbiol.28,226-231, 1974. For the purpose of comparison, the type culture ofActinomadura roseorufa ATCC 39,697 was used.

Identification media used for the characterization of the cultures andreferences for their composition are as follows:

1. Tryptone-Yeast Extract Broth--(ISP #1 medium, Difco).

2. Yeast Extract-Malt Extract Agar--(ISP #2 medium, Difco).

3. Oatmeal Agar--(ISP #3 medium, Difco).

4. Inorganic Salts-Starch Agar--(ISP #4 medium, Difco).

5. Glycerol-Asparagine Agar--(ISP #5 medium, Difco).

6. Peptone-Yeast Extract Iron Agar--(ISP #6 medium, Difco).

7. Czapek-Sucrose Agar--S. A. Waksman, The Actinomycetes, Vol. 2, mediumno. 1, p. 328, 1961.

8. Glucose-Asparagine Agar--Ibid, medium no. 2, p. 328.

9. Bennett's Agar--Ibid, medium no. 30, p. 331.

10. Emerson's Agar--Ibid, medium no. 28, p. 331.

11. Nutrient Agar--Ibid, medium no. 14, p. 330.

12. Gordon and Smith's Tyrosine Agar--R. E. Gordon and M. M. Smith, J.Bacteriol. 69: 147-150, 1955.

13. Casein Agar--Ibid.

14. Calcium Malate Agar--S. A. Waksman, Bacteriol. Rev. 21: 1-29, 1957.

15. Gelatin--R. E. Gordon and J. M. Mihm, J. Bacteriol. 73: 15-27, 1957.

16. Starch--Ibid.

17. Organic Nitrate Broth--Ibid.

18. Dextrose Nitrate Broth--S. A. Waksman, The Actinomycetes, Vol. 2,medium no. 1, p. 328, 1961, with 3 g dextrose substituted for 30 gsucrose and agar omitted.

19. Potato Carrot Agar--M. P. Lechevalier, J. Lab. and Clinical Med. 71:934-944, 1968, but use only 30 g potatoes, 2.5 g carrots and 20 g agar.

20. 2% Tap Water Agar.

21. Gauze's #1 Mineral Agar--G. F. Gauze et al., Problems in theClassification of Antagtonistic Actinomycetes, English Ed., p. 13, 1957.

22. Gauze's #2 Organic Agar--Ibid.

23. Skim Milk--Difco.

24. Cellulose utilization--

a) H. L. Jensen, Proc. Linn. Soc. N.S.W. 55: 1-248, 1930.

b) M. Levine and H. W. Schoenlein, A Compilation of Culture Media,medium no. 2511, 1930.

25. Utilization of Organic Acids--R. E. Gordon et al., Int. J. Syst.Bacteriol. 24: 54-63, 1974.

26. Acid Production from Carbohydrates--Ibid.

27. Hydrolysis of Hippurate and Esculin--Ibid.

28. Decomposition of Adenine, Hypoxanthine, Xanthine, and Urea--Ibid.

29. Resistance to Lysozyme--Ibid.

30. Carbohydrate Utilization--C-2 Medium, H. Nonomura and Y. Ohara, J.Ferment. Technol. 49: 887-894, 1971.

31. Temperature Range--ATCC medium 172 in ATCC Culture CollectionCatalogue, 15th ed., p. 608, 1982.

A Description of Culture FD-27684

Yeast Extract-Malt Extract Agar--Growth good, pink-red to red (6 1/2ia,7ia, 6ia), raised, wrinkled, with white aerial mycelium; reverse red(7ia); no soluble pigment.

Oatmeal Agar--Growth moderate, cream (2ca) , slightly raised, smooth, orappearing as isolated colonies; aerial mycelium none to sparse, white;reverse cream (2ca); no soluble pigment.

Inorganic Salts-Starch Agar--Growth poor to moderate, colorless to cream(2ca), thin, smooth; aerial mycelium none to sparse, white; reverse sameas surface; no soluble pigment.

Glycerol-Asparagine Agar--Growth poor to moderate, cream (2ca), withpink to red dots (6ea, 6 1/2ga); aerial mycelium none to sparse, white;reverse colorless to cream (2ca) , with red dots; no soluble pigment.

Czapek-Sucrose Agar--Growth poor to moderate, cream (2ca), with pink tored dots (5ea, 6 1/2ia); aerial mycelium none to sparse, white; reversecolorless to cream (2ca); no soluble pigment.

Glucose-Asparagine Agar--Growth moderate to good, pink to red (6 1/2ga,6 1/2na), raised; smooth, granular to wrinkled; aerial mycelium white topale pink (6ea); reverse red (6 1/2ga, 6 1/2ia); soluble pigment paleyellowish (3ca).

Gordon and Smith's Tyrosine Agar--Growth moderate to good, pink-orange(5ea), moderately raised, wrinkled; aerial mycelium none to sparse,white; reverse same as surface; soluble pigment yellowish (21c).

Calcium Maleate Agar--Growth scant, colorless, thin, smooth, no aerialmycelium; reverse colorless; no soluble pigment.

Casein Agar--Growth moderate to good, pink-orange to orange (4ia, 5ia),moderately raised, wrinkled, no aerial mycelium; reverse yellowish topale pink (3ga, 5ca); with brown (31c) soluble pigment.

Bennett's Agar--Growth good, red to dark red (6 1/2ne, 6 1/2ng), raised,wrinkled; aerial mycelium white to pink (6ea); reverse red (6 1/2lc);with brown (3ne) soluble pigment.

Emerson's Agar--Growth good to excellent, orange (51a, 5na), raised,wrinkled, with white aerial mycelium; reverse orange (5ic); no solublepigment.

Nutrient Agar--Growth moderate, pale orange (5ea, 5ga), slightly raised,smooth, or appearing as isolated colonies, no aerial mycelium; reversepale orange (5ga); no soluble pigment.

Gelatin Agar--Growth moderate to good, pale orange (4ga), moderatelyraised, smooth to wrinkled; aerial mycelium sparse, white; reverse paleorange (4ga); no soluble pigment.

Starch Agar--Growth moderate to good, pale orange (5ga), moderatelyraised, smooth to wrinkled; aerial mycelium sparse, white; reverse sameas surface; no soluble pigment.

Potato Carrot Agar--Growth poor to moderate, cream to pale pink (2ca,4ca), thin to slightly raised; aerial mycelium sparse, white; reversecream to pale pink (4ca); no soluble pigment.

Tap water Agar--Growth poor, colorless to cream (1 1/2ca), thin, smooth;aerial mycelium sparse, white; reverse same as surface; no solublepigment.

Gauze's Mineral Medium 1--Growth moderate, pink to red (5ca, 61a), withred dots (61c), slightly raised, smooth; aerial mycelium none to sparse,white; reverse same as surface; no soluble pigment.

Gauze's Organic Medium 2--Growth moderate to good, pink-orange (5ga),moderately raised, slightly wrinkled; aerial mycelium sparse, white;reverse same as surface; no soluble pigment.

Morphological Properties--After seven weeks of incubation, no sporeswere found on any of the media used. On potato carrot agar, however,hyphal swellings were produced terminally, laterally or intercalarily;and were single and smooth. They were globose, oval to elliptical, andmeasured 1.2-2.5 m diam. or 1.2-2.2×0.9-1.8 m. The similar structureswere also found on yeast extract-malt extract agar, oatmeal agar, tapwater agar, gelatin agar, Czapek-sucrose agar, and Gauze's mineralmedium 1.

Biochemical Properties--Melanin not produced; hydrogen sulfide notproduced; gelatin liquefied; starch not hydrolyzed; nitrate reduced tonitrite; slight growth on Jensen's cellulose broth but no growth onLevine and Schoenlein's cellulose broth; no disintegration on bothcellulose broths; coagulation and peptonization on milk; digestion ofcalcium malage negative; tyrosine digestion positive; caseindigestion-positive.

Carbohydrate utilization: glucose, rhamnose, and sucrose utilized;arabinose, fructose, inositol, mannitol, raffinose, and xylose notutilized.

The positive tests included: utilization of acetate, propionate, andpyruvate; acid production from glucose, rhamnose, maltose, andtrehalose.

The following tests were negative: decomposition of adenine, xanthine,hypoxanthine, and urea; hydrolysis of esculin and hippurate; resistanceto lysozyme; utilization of benzoate, citrate, dextrin, lactate, malate,mucate, oxalate, phenol, and succinate; acid production from arabinose,fructose, inositol, mannitol, raffinose, sucrose, xylose, adonitol,cellobiose, dulcitol, erythritol, galactose, glycerol, lactose, mannose,melezitose, malibiose, alpha-methyl-D-glucoside, ribose, salicin,sorbitol, sorbose, and starch.

Whole-cell Analysis--The whole-cell hydrolysates containmesodiaminopimelic acid, galactose, glucose, madurose, ribose, andrhamnose.

Temperature

    ______________________________________                                        21° C.                                                                             28° C.                                                                            37° C.                                                                            45° C.                               ______________________________________                                        Moderate    Good       Moderate   No                                          Growth      Growth     Growth     Growth                                      ______________________________________                                    

Culture FD-27684 is characterized by the inability to produce melanin;the pink, pink-orange, orange to red substrate mycelium; and thepresence of meso-diaminopimelic acid and madurose as whole-cellcomponents. Despite a long incubation period of up to seven weeks, theculture failed to produce spores although hyphal swellings were producedon some media. It is assignable to the genus Actinomadura.

Culture FD-27684 was similar to Actinomadura roseorufa Huang ATCC 39,697(see European Patent Application 169 001) in most of the culturalcharacteristics and almost all of the biochemical properties. On gelatinagar and starch agar, colonies of FD-27684 were pale orange rather thanpale cream. On tyrosine agar and Emerson's agar, they showed some tintof orange rather than brown. Culture FD-27684, unlike A. roseorufa,coagulated milk. These differences were minor and hence culture FD-27684is considered as a new strain of A. roseorufa.

Compared with the parent culture FD-27684, mutant FD-28474 produced lessaerial mycelium on yeast extract-malt extract agar, Bennett's agar,Gauze's organic medium 2, gelatin agar, and starch agar. Colonies of themutant were cream rather than orange on Emerson's agar and were palepink rather than cream to pale pink on potato carrot agar. The mutant,unlike its parent, produced hydrogen sulfide. All of the other culturalcharacteristics and biochemical properties were identical. Thus, mutantFD-28474 is considered as a new strain of Actinomadura roseorufa.

Compared with culture FD-28474 from which it was derived, mutantFD-28499 shared almost all of the cultural characteristics and all ofthe biochemical properties. The mutant differed from culture FD-28474only in the dark brown rather than dark red colonies on yeastextract-malt extract agar and in the presence of some pink dots onGauze's organic medium 2. Thus, mutant FD-28499 is considered as a newstrain of Actinomadura roseorufa.

FD-28454 was not subjected to taxonomic study. However, since it wasderived from a strain of Actinomadura roseorufa, and by mutationproduced a strain of Actinomadura roseorufa, it is considered to be astrain of Actinomadura roseorufa.

Antibiotic UK-61,689 exhibits inhibitory action against the growth of anumber of Gram-positive microorganisms. In Table I, below, the resultsof in vitro tests are summarized. For this test each organism isinoculated in a series of test tubes containing nutrient medium andvarying concentrations of Antibiotic UK-61,689 to determine the minimalconcentration of the compound in g/ml which inhibits the growth of theorganism over a period of 24 hours (MIC).

                  TABLE I                                                         ______________________________________                                        Antibacterial Activity                                                        Organism          Strain No. MIC μg/ml                                     ______________________________________                                        Clostridium perfringens                                                                         10A006     50                                                                 10A009     12.5                                             Actinomyces pyogenes                                                                            14D002     50                                                                 14D008     50                                                                 14D011     50                                               Treponema hyodysenteriae                                                                        94A001     3.12                                                               94A002     3.12                                                               94A007     1.56                                                               94A008     1.56                                             ______________________________________                                    

Efficacy data for UK-61,689 and its salts against coccidial infectionsin chickens were obtained in the following fashion. Groups of 3-5ten-day-old, pathogen-free white leghorn cockerel chicks were fed a mashdiet containing UK-61,689 or its sodium and/or potassium salt uniformlydispersed therein. After being on this ration for 24 hours, each chickwas inoculated per os with oocysts of the particular species of Eimeriabeing tested. Other groups of 3-5 ten-day-old chicks were fed a similarmash diet without Antibiotic UK-61,689 or its salts. They were alsoinfected after 24 hours and served as infected controls. Yet anothergroup of 3-5 ten-day-old chicks were fed the same mash diet withoutantibiotic UK-61,689 and were not infected with coccidia. These servedas normal controls. The results of treatment were evaluated after fivedays in the case of E. acervulina, and six days for all otherchallenges.

The criteria used to measure anticoccidial activity consisted of lesionscores of 0 to 4 for E. tenella after J. E. Lynch, "A New Method for thePrimary Evaluation of Anticoccidial Activity," Am. J. Vet. Res., 22,324-326, 1961; and 0 to 3 for the other species based on modification ofthe scoring system devised by J. Johnson and W. H. Reid, "AnticoccidialDrugs. Lesion Scoring Techniques in Battery and Floor Pen Experiments inChicks," Exp. Parasit., 28, 30-36, 1970. A constant ratio wasestablished by dividing the lesion score of each treated group by thelesion score of the infected control.

UK-61,689 and its cationic salts exhibit excellent activity againstcoccidial infections in poultry. When incorporated into the diet ofchickens at levels of 15 to 120 ppm, these compounds are effective incontrolling infections due to Eimeria tenella, E. acervulina, E. maxima,E. brunetti and E. necatrix.

For use in the treatment of coccidiosis in poultry the compound of thisinvention is administered orally in a suitable carrier. Conveniently,the medication is simply carried in the drinking water or in the poultryfeed, so that a therapeutic dosage of the agent ingested with the dailywater or poultry ration. The agent can be directly metered into drinkingwater, preferably in the form of a liquid, water-soluble concentrate(such as aqueous solution of a water soluble salt) or added directly tothe feed, as such, or in the form of a premix or concentrate. A premixor concentrate of therapeutic agent in a carrier is commonly employedfor the inclusion of the agent in the feed. Suitable carriers are liquidor solid, as desired, such as water, various meals; for example, soybeanoil meal, linseed oil meal, corncob meal, and mineral mixes such as arecommonly employed in poultry feeds. A particularly effective carrier isthe poultry feed itself; that is, a small portion of poultry feed.Further, the mycelium can be used as the carrier. The carrierfacilitates uniform distribution of the active materials in the finishedfeed with which the premix is blended. This is important because onlysmall proportions of the present potent agents are required. It isimportant that the compound be thoroughly blended into the premix and,subsequently, the feed. In this respect, the agent may be dispersed ordissolved in a suitable oily vehicle such as soybean oil, corn oil,cottonseed oil, and the like, or in a volatile organic solvent and thenblended with the carrier. It will be appreciated that the proportions ofactive material in the concentrate are capable of wide variation sincethe amount of agent in the finished feed may be adjusted by blending theappropriate proportion of premix with the feed to obtain a desired levelof therapeutic agent.

High potency concentrates may be blended by the feed manufacturer withproteinaceous carrier such as soybean oil meal and other meals, asdescribed above, to produce concentrated supplements which are suitablefor direct feeding to poultry. In such instances, the poultry arepermitted to consume the usual diet. Alternatively, such concentratedsupplements may be added directly to the poultry feed to produce anutritionally balanced, finished feed containing a therapeuticallyeffective level of the compound of this invention. The mixtures arethoroughly blended by standard procedures, such as in a twin shellblender, to ensure homogeneity.

It will, of course, be obvious to those skilled in the art that the uselevels of the compound described herein will vary under differentcircumstances. Continuous low-level medication, during the growingperiod; that is, during the first 6 to 12 weeks for chickens, is aneffective prophylactic measure. In the treatment of establishedinfections, higher levels may be necessary to overcome the infection.The use level in feed will generally be in the range of 15 to 120 ppm.When administered in drinking water, the level which will be that whichwill provide the same daily dose of medication, i.e., 15 to 120 ppm,factored by the weight ratio of the average daily consumption of feed tothe average daily consumption of water.

The present invention is illustrated by the following examples. However,it should be understood that the invention is not limited to thespecific details of these examples.

EXAMPLE 1

A. Preparation of Inoculum

A sterile aqueous medium having the following composition was prepared.

    ______________________________________                                        Ingredient      Grams/liter                                                   ______________________________________                                        Cerelose        10.0                                                          Corn starch     5.0                                                           Corn steep liquor                                                                             5.0                                                           NZ Amine YTT*   5.0                                                           Cobalt chloride 0.002                                                         ______________________________________                                         *(Registered trademark for enzymatic digest of casein, Humko Sheffield        Chemical Co. Inc.)                                                       

After the pH was adjusted to 7.0, the medium was dispensed (800 ml) into2800 ml Fernbach flasks, cotton plugged/paper capped and sterilized byautoclaving for 60 minutes at 121° C. (15 p.s.i.). After cooling, themedium was inoculated with a vegetative cell suspension from a slant ofFD-28454 (ATCC 53,674). The flasks were shaken at 28° C. on a rotaryshaker having a displacement of 1 1/2 to 2 1/2 inches and 150 to 200cycles per minute for 6 days.

B. Fermentation and Isolation of UK-61,689.

A Fernbach flask containing 800 ml of the grown culture was used toinoculate a 14-liter fermentation vessel containing 8 liters of sterilemedium of the following composition to which 4 ml of siliconeanti-foaming agent had been added:

    ______________________________________                                        Ingredient        Grams/liter                                                 ______________________________________                                        Cerelose          45.0                                                        Soy flour         10.0                                                        Corn steep liquor 15.0                                                        Blood meal        5.0                                                         Corn flour        5.0                                                         MnSO.sub.4.H.sub.2 O                                                                            0.1                                                         MgSO.sub.4.7H.sub.2 O                                                                           0.1                                                         COCl.sub.2.6H.sub.2 O                                                                           0.002                                                       Calcium carbonate 3.0                                                         pH adjusted to 6.9-7.0                                                        ______________________________________                                    

Fermentation was carried out at 30° C. with stirring at 500 revolutionsper minute and aeration at 0.75 volume air per volume of broth perminute until substantial activity was produced. The UK-61,689/UK-58,852in the broth and recovery streams was visualized by using silica gelthin layer chromatography plates Developed with a system consisting of9:1 chloroform:methanol. The plates were sprayed with vanillin reagent(6 g vanillin in 100 ml ethanol and 3% concentrated H₂ SO₄) and heatedat 100° C. for 5 minutes. UK-61,689 appears as a reddish-blue spot.Alternatively, the plate was overlayed with agar seeded with B.subtilis, to which 0.4 ml of a 5% solution of2,3,5-triphenyl-2H-tetrazolium chloride had been added, and incubated at37° C. for 16 hours to visualize the antibiotic as a colorless areaagainst a red background.

The whole broth was then extracted with methylisobutyl ketone and thesolvent concentrated to yield 14.4 g residue. This material waschromatographed on a 6×100 cm column packed with column grade silica gelG (70-230 mesh, Woelm) in ethyl acetate. The column was developed withethyl acetate at a flow rate of ˜20 ml/min. Fractions of 10 ml each weretaken.

The fractions were examined by thin-layer chromatography on Analtechsilica gel GF plates developed with 9 CHCl₃ :1 MEOH and visualized byspraying with vanillin reagent and heating.

The fractions containing antibiotic UK-61,689 were combined (totalvolume approximately 200 ml) and stirred with ˜2 g Darco G60 for 15minutes. After removing the carbon by filtration, the filtrate (ethylacetate) was washed with dibasic sodium-phosphate, 5% buffer adjusted topH 10.0 with 1N NaOH. After separation, the ethyl acetate layer wasdried over anhydrous sodium sulfate and then evaporated under vacuum.The viscous oil remaining after evaporation was dissolved in a smallvolume of heptane whereupon crystallization occurred. The crystals werecollected by filtration and dried under vacuum yielding 1.4 g ofantibiotic UK-61,689 as the sodium salt; m p. 167° C.; C¹³ NMR (CDCl₃)in ppm: 179.16, 107.54, 103.21, 97.80, 97.01, 87.02, 84.65, 84.28,82.39, 82.10, 80.92, 80.28, 79.96, 77.62, 77.11, 76.60, 74.91, 74.65,73.15, 70.19, 67.74, 66.94, 59.07, 56.84, 45.49, 39.92, 39.00, 36.55,33.88, 33.79, 33.63, 33.51, 33.21, 32.51, 32.33, 30.63, 27.64, 26.98,26.91, 26.16, 23.25, 18.43, 17.53, 17.00, 12.13, 11.05, 10.47.

The fractions containing UK-58,852 are combined, treated with ˜2 g DarcoG60 as above, then concentrated. The residue is suspended in hexane andbatch treated with silica gel on a filter funnel. The absorbant iswashed with hexane, then eluted with chloroform and ethyl acetate. Theethyl acetate fraction is concentrated, the residue rechromatographed onsilica and the product crystallized from heptane to give antibioticUK-58,852 as a white solid.

EXAMPLE 2

The procedure of Example 1 was followed but using FD-28499 (ATCC 53,664)in place of FD-28454 (ATCC 53,674). HPLC of the methyl isobutyl ketoneextract of the whole broth afforded UK-61,689 substantially free (<1%)of UK-58,852. Its TLC behavior was identical to that reported in Example1 for UK-61,689.

The acid form of UK-61,689 is prepared by stirring a chloroform solutionof the sodium salt with an equal volume of water and lowering the pH to3.0 with phosphoric acid. The phases are then separated, and thechloroform evaporated under vacuum to give Antibiotic UK-61,689 as thefree acid.

EXAMPLE 3

FD-28474 (ATCC 53,665) was fermented according to the procedure ofExample 1 except the fermentation medium contained no corn steep liquoror blood meal. The whole broths from four such fermentations werecombined and filtered. The filtrate and mycelium were each extractedwith methyl isobutyl ketone (3×200 ml). The extracts were combined andconcentrated under reduced pressure to an oil (18.5 g). The oil wastaken up in acetone (200 ml) and the solution divided into two equalvolumes (I and II).

The pH of volume I was adjusted to 12 by addition of aqueous sodiumhydroxide (20%). The alkaline solution was then filtered andconcentrated to an oil under reduced pressure. Actone (10 ml), heptane(79 ml) and water (39 ml) were added to the oil to provide dark browncrystals. Repulping of the crystals in heptane gave light brown crystals(2 g) comprising 75% UK-61,689 and 5% UK-58,852 by HPLC assay.

Volume 2 was concentrated to an oil under reduced pressure and the oildissolved in ethyl acetate (100 ml). It was then subjected to columnchromatography over silica gel (500 g) using ethyl acetate as elutingagent. The UK-61,689 rich fractions were combined and concentrated to anoil. The oil was taken up in acetone (10 ml)-heptane (110 ml) and theresulting crystals of UK-61,689 filtered and dried (1.2 g). TheUK-58,852 was not recovered.

I claim:
 1. A process for making the compound UK-61,689 having theformula (I) ##STR3## which comprises fermenting, in an aqueous mediumcontaining assimilable sources of carbon, nitrogen and inorganic saltsunder submerged aerobic conditions, a strain belonging to the genusActinomadura, wherein said strain is a mutant obtained by mutating aUK-58,852 producing strain of Actinomadura roseorufa and which mutantproduces UK-61,689 upon cultivation in said aqueous nutrient medium,until a recoverable amount of compound UK-61,689 accumulates in thewhole broth; and recovering compound UK-61,689.
 2. A process accordingto claim 1 wherein the compound of formula (I) is recovered from thefermentation broth in association with mycelium.
 3. A process for makingthe compound UK-61,689 having the formula (I) ##STR4## which comprisesfermenting Actinomadura roseorufa which produces compound UK-61,689 inan aqueous nutrient medium containing assimilable sources of carbon,nitrogen and inorganic salts under submerged aerobic fermentationconditions until a recoverable amount of compound UK-61,689 accumulatesin the whole broth; and recovering compound UK-61,689.
 4. A processaccording to claim 3 wherein the compound of formula (I) is recoveredfrom the fermentation broth in association with mycelium.
 5. A processaccording to claim 3 which comprises cultivating Actinomadura roseorufaATCC 53,665 or ATCC 53,674.
 6. A process according to claim 3 whichcomprises cultivating Actinomadura roseorufa ATCC 53,664.